Apolipoprotein A-IV inhibits experimental colitis (original) (raw)

Animals. Male C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA). Male ApoA-IV–/– (51) animals were originally obtained from J.L. Breslow and were backcrossed onto the parent C57BL/6J strain for eight generations. Animals were maintained on a 12-hour light/12-hour dark cycle under pathogen-free conditions. The mice had ad libitum access to a standard diet and water until reaching the desired age (8–10 weeks) and/or weight (20–25 g). All procedures using animals were reviewed and approved by the Institutional Animal Care and Use Committee of Louisiana State University Health Sciences Center and were performed according to the criteria outlined by the NIH.

Recombinant human apoA-IV. We produced rhA-IV using the apoA-IV expression vector pL2102-hA-IV, modified from pL1867-hA-IV, which was previously described by Duverger et al. (52). This construct encodes the full-length protein–coding portion of the apoA-IV gene, with a 6X-histidine tag to facilitate purification. rhA-IV from this construct was previously demonstrated to be physicochemically and functionally similar to native human apoA-IV purified from plasma (52). rhA-IV was produced in BL21(DE3)pLysS competent cells (Promega Corp., Madison, Wisconsin, USA) after transformation with pL2102-hA-IV. After overnight culture, cells were diluted 1:4 with fresh media and allowed to grow to an OD610 of 0.5, then induced for 2 hours with 1 mM isopropyl β-D-thiogalactoside. Cells were harvested and washed, then lysed in 20 mM PBS (19.7 mM NaH2PO4, 0.26 mM Na2HPO4, and 500 mM NaCl, pH 7.8) by freeze-thawing followed by sonication. Bacterial lysates were incubated in batchwise fashion with Ni2+-charged agarose resin (Pro-Bond; Invitrogen Corp., Carlsbad, California, USA), then poured into an 18 cm × 2 cm column. The column was washed extensively with 20 mM PBS, followed by 20 mM PBS plus 40 mM imidazole; completeness of washing was monitored by continuous measurement of OD280 and by SDS-PAGE on selected fractions. rhA-IV was eluted from the column free of contaminating bacterial proteins using 20 mM PBS plus 150 mM imidazole. Eluted rhA-IV was dialyzed exhaustively against PBS (pH 7.4), concentrated against PEG 8000, filter-sterilized, and then stored in small aliquots at –80°C until needed. Freshly thawed rhA-IV was used for all experiments.

rhA-I. rhA-I was produced as previously described (53), lyophilized, and dissolved in PBS injection vehicle immediately prior to administration. The injected molar dose of rhA-I (0.31 mg/d) was equivalent to rhA-IV at 0.5 mg/d.

Induction of colitis. Mice were administered 3% DSS (mol wt, 40 kDa; ICN Biomedicals, Aurora, Ohio, USA) dissolved in water that was filter-purified (Millipore Corp., Bedford, Massachusetts, USA) for 7 days (day 0–6 ad libitum) (26). Control C57BL/6J mice received the filtered water alone.

Assessment of inflammation in DSS-treated mice. Clinical assessment of mice included daily measurement of body weight, evaluation of stool consistency, and the presence of blood in the stools by a guaiac paper test (ColoScreen; Helena Laboratories Inc., Beaumont, Texas, USA) (54). Clinical DAI (25) ranging from 0 to 4 was calculated using the following parameters: stool consistency (normal, loose, diarrhea), presence or absence of fecal blood (guaiac paper test and macroscopic evaluation of the anus), and weight loss. On day 6, intravital microscopy was performed and the mice were sacrificed. Colons were removed, from the cecum to the pelvic floor. Colon length and weight were measured before dividing the colon for histology and evaluation of MPO activity.

DSS load calculation. Previously, we demonstrated that in DSS-induced colitis, the severity of inflammation is DSS load–dependent, and that a critical DSS load greater than or equal to 30 mg DSS/g body wt is required to reliably induce colitis in the C57BL/6J strain (29). DSS load for all DSS-treated mice was calculated as: load = [total drinking volume (ml) × DSS (g) per 100 ml]/initial body wt (g). The drinking volume was recorded daily using calibrated water bottles.

Histology. For each animal, histological examination was performed on three samples of the distal colon; samples were fixed in 10% formalin before staining with H&E. All histological quantitation was performed blinded using a scoring system previously described (30). The three independent parameters measured were severity of inflammation (0–3: none, slight, moderate, severe), extent of injury (0–3: none, mucosal, mucosal and submucosal, transmural), and crypt damage (0–4: none, basal 1/3 damaged, basal 2/3 damaged, only surface epithelium intact, entire crypt and epithelium lost). The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement (×1: 0–25%, ×2: 26–50%, ×3: 51–75%, ×4: 76–100%) and all numbers were summed. Maximum possible score was 40.

For determination of lamina propria leukocytes, H&E-stained 5-μm sections were examined under high-power magnification (×25). Neutrophils in the lamina propria were counted in five randomly selected high-power fields on each slide and the average per slide was determined.

Tissue MPO activity. MPO was measured in tissue from proximal to distal colon (adjacent to tissue used for histology). Samples were rinsed with cold PBS, blotted dry, and immediately frozen in liquid nitrogen. They were stored at –80°C until assayed for MPO activity using the _o_-dianisidine method (55, 56). Tissue samples were thawed and weighed. The samples were then suspended (10% wt/vol) in 50 mM pH 6.0 potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (0.1 g/20 ml potassium phosphate) and homogenized. A sample of the homogenate (1 ml) was sonicated for 30 seconds, then centrifuged at 200 g for 10 minutes at 4°C. The reaction was begun by mixing and incubating the supernatant (100 μl) at 20°C for 10 minutes with a solution composed of 2,810 μl of 50 mM potassium phosphate, 30 μl of 20 mg/ml _o_-dianisidine dihydrochloride, and 30 μl of 20 mM hydrogen peroxide. After 10 minutes the reaction was terminated by the addition of 30 μl 2% sodium azide. The change in absorbance was read at 460 nm in a spectrophotometer (UV-1201 Series; Shimadzu Scientific Instruments Inc., Kyoto, Japan). MPO activity was expressed as the amount of enzyme necessary to produce a change in absorbance of 1.0 unit per minute per g tissue (wet weight).

Blood sampling and platelet preparation. Platelets were derived from untreated C57BL/6J mice for all intravital experiments. Donor mice were anesthetized using ketamine hydrochloride (150 mg/kg intraperitoneally) and xylazine (7.5 mg/kg intraperitoneally). As described previously (57), approximately 0.9 ml of blood was harvested via a catheter placed in the carotid artery. The blood was collected in polypropylene tubes containing 0.1 ml acid-citrate-dextrose buffer (Sigma-Aldrich, St. Louis, Missouri, USA). Platelet-rich plasma was obtained by two sequential centrifugations (120 g for 8 minutes and 120 g for 3 minutes). The platelet-rich plasma was removed and centrifuged again at 550 g for 10 minutes, and the pellet was resuspended in PBS, pH 7.4. Platelets were then incubated for 10 minutes at room temperature with the fluorochrome CFSE (90 mM final concentration; Molecular Probes Inc., Eugene, Oregon, USA). The fluorescently labeled platelet solution was then centrifuged and the pellet was resuspended in 500 μl of PBS and protected from light until infused into a recipient animal. Leukocytes accounted for 0.01% of the cells in the platelet suspension. The number of fluorescently labeled platelets obtained from one donor mouse was sufficient for two recipient mice.

Surgical preparation for intravital microscopy. Animals were anesthetized as described above. The right carotid artery was cannulated for blood pressure measurements using a disposable pressure transducer (Cobe Laboratories Inc., Lakewood, Colorado, USA) attached to a pressure monitor (BP-1; World Precision Instruments, Sarasota, Florida, USA). The right jugular vein was cannulated for infusion of rhodamine 6G (Sigma-Aldrich) for leukocyte labeling and subsequent infusion of CFSE-labeled platelets. On an adjustable acrylic microscope stage, a laparotomy was performed and the animal was placed on its right side. The proximal large bowel (initial 2–3 cm adjacent to the cecum) was exteriorized with moist cotton swabs, covered with a nonwoven sponge, and superfused at 37°C with bicarbonate-buffered saline solution (pH 7.4).

Intravital fluorescence microscopy. Platelets and leukocytes were visualized with an inverted Nikon microscope (Nikon Inc., Tokyo, Japan) equipped with a 75-watt XBO xenon lamp. Visualization of CFSE (excitation: 490 nm, emission: 518 nm) and rhodamine 6G (excitation: 525 nm, emission 550 nm) required a Nikon filter block with an excitation filter (470–490 nm), a dichroic mirror (510 nm), and a barrier filter (520 nm). With a ×40 objective (0.85 numerical aperture, Nikon Inc.), the magnification on the television screen (Trinitron PVM-2030, 50.6 cm diagonal; Sony Corp., Tokyo, Japan) was ×1,280. The microscopic images were received by a charge-coupled device (CCD) video camera (model C2400; Hamamatsu Photonics KK, Hamamatsu, Shizuoka, Japan) and optimized by a CCD camera controller (model C2400; Hamamatsu Photonics KK). The images were recorded on a videocassette recorder (HS-U65; Mitsubishi Digital Electronics America, Irvine, California, USA). A video time-date generator (Panasonic WJ810; Matsushita Electric Industrial Co., Osaka, Japan) projected the time, date, and stopwatch function onto the monitor. Five randomly chosen postcapillary venules of the proximal large bowel were each recorded for 1 minute.

Video analysis. Venular diameter (between 20 μm and 40 μm) was measured with a video caliper (Microcirculation Research Institute, Texas A&M University, College Station, Texas, USA) and venular length was set at 100 μm. Platelets and leukocytes were classified according to their interaction with the venular wall as either free flowing or adherent (when cells remained stationary for 30 seconds or more). We determined whether a platelet or leukocyte was adherent directly to the endothelium itself or indirectly by attachment to another blood cell, suggesting binding of blood cells to other blood cells and to endothelium. Platelet and leukocyte adherence was expressed as the number of cells per mm2 of venular surface, calculated from diameter and length, assuming cylindrical vessel shape.

Measurement of in vivo colonic P-selectin expression using dual radiolabeled mAb technique. The binding mAb’s used for the in vivo characterization of P-selectin expression were RB40.34, a rat immunoglobulin (IgG1) that is specific for mouse CD62P (P-selectin) (BD Biosciences — Pharmingen, San Diego, California, USA), and P23, a nonbinding murine IgG1 directed against human P-selectin (Pharmacia-Upjohn, Kalamazoo, Michigan, USA). The binding (RB40.34) and nonbinding (P23) mAb’s were labeled with 125I and 131I (DuPont NEN Research Products, Boston, Massachusetts, USA), respectively, using the iodogen method as described previously (58), and stored at 4°C. Mice were anesthetized as described above, then equipped with right jugular and carotid catheters. A mixture (200-μl) of 125I-labeled binding mAb and 131I-labeled nonbinding mAb was administered through the jugular vein catheter. Five minutes after injection of the mAb mixture, a blood sample was obtained from the carotid artery. Immediately thereafter, the animal was rapidly exsanguinated by jugular perfusion of bicarbonate-buffered saline, immediately followed by carotid perfusion with bicarbonate-buffered saline after severing the inferior vena cava at the thoracic level. The large bowel was harvested and divided into proximal and distal portions. The method for calculating P-selectin expression has been described previously (58). Briefly, activity of 125I and 131I (marking the binding mAb and the nonbinding mAb, respectively) in the tissue and in 50-μl samples of cell-free plasma was counted in a 14800 Wizard 3 γ counter (Wallac, Turku, Finland). The accumulated activity of the labeled mAb in the colon was expressed as the percentage of the injected activity per g tissue. P-selectin expression was calculated by subtracting the accumulated activity per g tissue of the nonbinding mAb from the activity of the binding P-selectin–binding mAb. This value, expressed as percent injected dose per g tissue, was converted to ng mAb per g tissue by multiplying the above value by the total injected binding mAb.

Experimental protocols. In the first series of experiments, we tested different doses of intraperitoneally administered rhA-IV (0.5 mg and 0.25 mg daily, n = 7 each) in DSS-induced colitis starting at day 0 for 7 days. Control C57BL/6J mice (n = 7) received water. Besides animals receiving DSS (n = 7), a vehicle control group received DSS + PBS intraperitoneally (n = 4). To control for nonspecific protein effects, two more DSS groups received BSA (n = 3) and rhA-I (n = 6) (both in molar dose equivalents to 0.5 mg rhA-IV/d), respectively.

In the second series of experiments we examined the effect of rhA-IV on colonic microvasculature using intravital fluorescence microscopy. For 7 days, C57BL/6J mice received either water (n = 6), DSS (n = 6), or DSS + rhA-IV (0.5 mg/d) (n = 6) until microscopy was performed. One hundred microliters of 0.02% rhodamine 6G was infused intrajugularly over 5 minutes and allowed to circulate for 5 minutes. Immediately thereafter, fluorescently labeled platelets (100 × 106) were infused over a period of 5 minutes using the infusion pump and then allowed to circulate for 5 minutes before beginning recording.

In a third series of experiments we tested whether apoA-IV deficiency aggravates DSS-induced colitis. All animals in this third series of experiments were kindly provided by the laboratory of Patrick Tso. The experimental groups were: WT C57BL/6J mice treated with water (n = 5); WT C57BL/6J mice (littermates of ApoA-IV–/– mice) treated with DSS (n = 6); ApoA-IV–/– mice treated with DSS (n = 6); and ApoA-IV–/– mice treated with DSS + rhA-IV (0.5 mg/d, n = 6).

Data analysis. Statistical analyses were performed with StatView 4.5 software (Abacus Concepts Inc., Berkeley, California, USA) using one-way ANOVA followed by the Scheffé (post hoc) test. All values are reported as mean ± SE. Statistical significance was set at P < 0.05.